Local Complement Contributes to Pathogenic Activation of Lung Endothelial Cells in SARS-CoV-2 Infection.

Endothelial dysfunction and inflammation contribute to the vascular pathology of Coronavirus Disease 2019 (COVID-19). However, emerging evidence does not support direct infection of endothelial or other vascular wall cells and thus inflammation may be better explained as secondary responses to epithelial cell infection. In this study, we sought to determine whether lung endothelial or other resident vascular cells are susceptible to productive SARS-CoV-2 infection and how local complement activation contributes to endothelial dysfunction and inflammation in response to hypoxia and SARS-CoV-2 infected lung alveolar epithelial cells. We found that ACE2 and TMPRSS2 mRNA expression in lung vascular cells including primary human lung microvascular endothelial cells (HLMVEC), pericytes, smooth muscle cells and fibroblasts was 20-90-fold lower compared to primary human alveolar epithelial type II (AT2) cells. Consistently, we found that HLMVEC and other resident vascular cells were not susceptible to productive SARS-CoV-2 infection under either normoxic or hypoxic conditions. However, viral uptake without replication (abortive infection) was observed in HLMVEC when exposed to conditioned medium from SARS-CoV-2 infected human ACE2 stably transfected A549 epithelial cells (hACE2-A549). Furthermore, we demonstrated that exposure of HLMVEC to conditioned medium from SARS-CoV-2 infected hACE2-A549 cells and hypoxia resulted in upregulation of inflammatory factors such as ICAM1, VCAM1, IL-6 as well as complement components such as C3, C3AR1, C1QA and CFB. Taken together, our data support a model in which lung endothelial/vascular dysfunction during COVID-19 involves the activation of complement and inflammatory signaling and does not involve productive viral infection of endothelial cells. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

Magnetic Enrichment of SARS-CoV-2 Antigen-Binding B Cells for Analysis of Transcriptome and Antibody Repertoire

The ongoing COVID-19 pandemic has had devastating health impacts across the globe. The development of effective diagnostics and therapeutics will depend on the understanding of immune responses to natural infection and vaccination to the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While both B-cell immunity and T-cell immunity are generated in SARS-CoV-2-infected and vaccinated individuals, B-cell-secreted antibodies are known to neutralize SARS-CoV-2 virus and protect from the disease. Although interest in characterizing SARS-CoV-2-reactive B cells is great, the low frequency of antigen-binding B cells in human blood limits in-depth cellular profiling. To overcome this obstacle, we developed a magnetic bead-based approach to enrich SARS-CoV-2-reactive B cells prior to transcriptional and antibody repertoire analysis by single-cell RNA sequencing (scRNA-seq). Here, we describe isolation of SARS-CoV-2 antigen-binding B cells from two seropositive donors and comparison to nonspecific B cells from a seronegative donor. We demonstrate that SARS-CoV-2 antigen-binding B cells can be distinguished on the basis of transcriptional profile and antibody repertoire. Furthermore, SARS-CoV-2 antigen-binding B cells exhibit a gene expression pattern indicative of antigen experience and memory status. Combining scRNA-seq methods with magnetic enrichment enables the rapid characterization of SARS-CoV-2 antigen-binding B cells.

COVID-19 Serology Control Panel Using the Dried-Tube Specimen Method.

The dried-tube specimen (DTS) procedure was used to develop the COVID-19 serology control panel (CSCP). The DTS offers the benefit of shipping materials without a cold chain, allowing for greater access without deterioration of material integrity. Samples in the panel were sourced from COVID-19 convalescent persons from March to May 2020. The immunoglobulin subtypes (total Ig, IgM, and IgG) and their respective reactivity to severe acute respiratory syndrome coronavirus 2 nucleocapsid, spike, and receptor-binding domain antigens of the samples were delineated and compared with the WHO International Standard to elucidate the exact binding antibody units of each CSCP sample and ensure the CSCP provides adequate reactivity for different types of serological test platforms. We distribute the CSCP as a kit with five coded tubes to laboratories around the world to be used to compare test kits for external quality assurance, for harmonizing laboratory testing, and for use as training materials for laboratory workers.

Lipid nanoparticle formulation of niclosamide (nano NCM) effectively inhibits SARS-CoV-2 replication in vitro.

As exemplified by the COVID-19 pandemic, highly infective respiratory viruses can spread rapidly in the population because of lack of effective approaches to control viral replication and spread. Niclosamide (NCM) is an old anthelminthic drug (World Health Organization essential medicine list) with pleiotropic pharmacological activities. Several recent publications demonstrated that NCM has broad antiviral activities and potently inhibits viral replication, including replication of SARS-CoV-2, SARS-CoV, and dengue viruses. Unfortunately, NCM is almost completely insoluble in water, which limits its clinical use. We developed a cost-effective lipid nanoparticle formulation of NCM (nano NCM) using only FDA-approved excipient and demonstrated potency against SARS-CoV-2 infection in cells (Vero E6 and ACE2-expressing lung epithelium cells).

Development and Validation of a Multiplex Microsphere Immunoassay Using Dried Blood Spots for SARS-CoV-2 Seroprevalence: Application in First Responders in Colorado, USA.

Serological testing of large representative populations for antibodies to SARS-CoV-2 is needed to estimate seroprevalence, transmission dynamics, and the duration of antibody responses from natural infection and vaccination. In this study, a high-throughput SARS-CoV-2 multiplex microsphere immunoassay (MMIA) was developed for the receptor binding domain (RBD) and nucleocapsid (N) that was more sensitive than enzyme-linked immunosorbent assay (ELISA) (98% versus 87%). The MMIA was then applied and validated in 264 first responders in Colorado using serum and dried blood spot (DBS) eluates, compared to ELISA, and evaluated for neutralizing antibodies. Four percent (11/264) of first responders were seropositive in July to August 2020. Serum and DBS were highly correlated for anti-RBD and anti-N antibodies (R = 0.83, P < 0.0001 and R = 0.87, P < 0.0001, respectively) by MMIA. The MMIA accurately predicted SARS-CoV-2 neutralizing antibodies using DBS (R = 0.76, P = 0.037). On repeat antibody testing 3 months later, anti-RBD IgG decreased less rapidly than anti-N IgG measured by MMIA, with a median change in geometric median fluorescence intensity of 62% versus 79% (P < 0.01) for anti-RBD and anti-N IgG, respectively. This novel MMIA using DBS could be scalable for rapid and affordable SARS-CoV-2 serosurveillance in the United States and globally.

Mechanisms of SARS-CoV-2-induced lung vascular disease: potential role of complement.

The outbreak of COVID-19 disease, caused by SARS-CoV-2 beta-coronovirus, urges a focused search for the underlying mechanisms and treatment options. The lung is the major target organ of COVID-19, wherein the primary cause of mortality is hypoxic respiratory failure, resulting from acute respiratory distress syndrome, with severe hypoxemia, often requiring assisted ventilation. While similar in some ways to acute respiratory distress syndrome secondary to other causes, lungs of some patients dying with COVID-19 exhibit distinct features of vascular involvement, including severe endothelial injury and cell death via apoptosis and/or pyroptosis, widespread capillary inflammation, and thrombosis. Furthermore, the pulmonary pathology of COVID-19 is characterized by focal inflammatory cell infiltration, impeding alveolar gas exchange resulting in areas of local tissue hypoxia, consistent with potential amplification of COVID-19 pathogenicity by hypoxia. Vascular endothelial cells play essential roles in both innate and adaptive immune responses, and are considered to be "conditional innate immune cells" centrally participating in various inflammatory, immune pathologies. Activated endothelial cells produce cytokines/chemokines, dynamically recruit and activate inflammatory cells and platelets, and centrally participate in pro-thrombotic processes (thrombotic microangiopathies). Initial reports presented pathological findings of localized direct infection of vascular endothelial cells with SARS-CoV-2, yet emerging evidence does not support direct infection of endothelial or other vascular wall cell and thus widespread endothelial cell dysfunction and inflammation may be better explained as secondary responses to epithelial cell infection and inflammation. Endothelial cells are also actively engaged in a cross-talk with the complement system, the essential arm of innate immunity. Recent reports present evidence for complement deposition in SARS-CoV-2-damaged lung microcirculation, further strengthening the idea that, in severe cases of COVID-19, complement activation is an essential player, generating destructive hemorrhagic, capillaritis-like tissue damage, clotting, and hyperinflammation. Thus, complement-targeted therapies are actively in development. This review is intended to explore in detail these ideas.

A New SARS-CoV-2 Dual-Purpose Serology Test: Highly Accurate Infection Tracing and Neutralizing Antibody Response Detection.

Many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology tests have proven to be less accurate than expected and do not assess antibody function as neutralizing, correlating with protection from reinfection. A new assay technology measuring the interaction of the purified SARS-CoV-2 spike protein receptor binding domain (RBD) with the extracellular domain of the human angiotensin-converting enzyme 2 (hACE2) receptor detects these important antibodies. The cPass surrogate virus neutralization test (sVNT), compared directly with eight SARS-CoV-2 IgG serology and two live-cell neutralization tests, gives similar or improved accuracy for qualitative delineation between positive and negative individuals in a fast, scalable, and high-throughput assay. The combined data support the cPass sVNT as a tool for highly accurate SARS-CoV-2 immunity surveillance of infected/recovered and/or vaccinated individuals as well as drug and convalescent-phase donor screening. The data also preview a novel application for the cPass sVNT in calibrating the stringency of live-cell neutralization tests and its use in longitudinal testing of recovered and/or vaccinated patients.

Presence and short-term persistence of SARS-CoV-2 neutralizing antibodies in COVID-19 convalescent plasma donors.

BACKGROUND: In March 2020, the Food and Drug Administration (FDA) approved use of COVID-19 convalescent plasma (CCP) as an investigational new drug for treatment of COVID-19. Since then, collection of CCP from COVID-19-recovered patients has been implemented in donor centers nationwide. Children's Hospital Colorado rapidly put into practice a CCP collection protocol, necessitating development and implementation of assays to evaluate SARS-CoV-2 antibodies in CCP units. STUDY DESIGN AND METHODS: We evaluated 87 units of CCP collected from 36 donors over two to four sequential donations using both antigen-binding assays for SARS-CoV-2 nucleoprotein and spike antigens and a live virus focus reduction neutralization test (FRNT50 ). RESULTS: Our data show that the majority of donors (83%) had a FRNT50 titer of at least 80, and 61% had a titer of at least 160, which met the FDA's criteria for acceptable CCP units. Additionally, our data indicate that analysis of antibodies to a single SARS-CoV-2 antigen is likely to miss a percentage of seroconverters; however, these individuals tend to have neutralizing antibody titers of less than 80. There was considerable variability in the short-term, sustained antibody response, measured by neutralizing antibody titers, among our donor population. CONCLUSION: The correlation of neutralizing activity and antigen-binding assays is necessary to qualify CCP for therapeutic use. Since SARS-CoV-2 antibody levels decline in a percentage of donors, and such a decline is not detectable by current qualitative assays implemented in many laboratories, robust, quantitative assays are necessary to evaluate CCP units best suited for therapeutic infusion in COVID-19 patients.